The present invention relates to the treatment of pathological conditions associated with various endogenous peptides. In particular, the invention relates to the use of enkephalinase (E.C. 3.4.24.11) and novel forms thereof in such treatment.
Various endogenous peptides have been discovered which appear active in various physiological systems. For example, two pentapeptides, referred to as enkephalins, were extracted from the brain. The effects of enkephalins include analgesia, thermoregulation, tranquilization, gastrointestinal function and increasing appetite. Until the present invention, the sole physiological activity of enkephalinase was thought to be the cleavage of enkephalins in the central nervous system (Schwartz, J. C. et al., Trends Pharmacol Sci. 6: 472-476 [1985]). which to date is not known to be associated with any pathological disorder. Because such activity would result in an enhancement of pain, scientific research has focused on inhibiting enkephalinase. This invention establishes therapeutic uses of enkephalinase for the first time.
Another endogenous peptide, angiotensin II, is presumed to be an etiologic agent in the pathological condition of renal hypertension. Bradykinin and kallidin have been associated with other pathological conditions such as acute inflammation associated with burns, rheumatoid arthritis, edema, carcinoid syndrome, pancreatitis, migraine headache, reactions after transfusion with plasma products, allergic diseases, endotoxic shock and anaphylactic shock.
Another class of endogenous peptides are the tachykinins which share some of the same physiological activity. The tachykinins include substance P, eledoisin, neurokinin A and B, physalaemin and kassinin Substance P has been shown to be associated with smooth muscle contraction, neurotransmission, pain, cough, exocrine secretion, vasodilation, increased vascular permeability, increased adherence of leukocytes to venules, stimulation of polymorphonuclear leukocytes, macrophages, T lymphocytes, and degranulation of mast cells. Endogenous peptides such as bombesin have been found to be present in endocrine cells in normal lungs (Cutz et al., Experientia 37, 765-767 [1981]), released from carcinoid tumors, and implicated in the associated cutaneous flushes, telangiectasia, diarrhea and bronchoconstriction.
Bombesin functions as a growth factor for airway epithelial cells (Willey et al., Exp. Cell Res. 153, 245-248 [1984]) and for human small-cell lung cancer (Cuttitta, F. et al., Nature 316, 823-826 [1985]). Substance P and bombesin, apart from their tumor-associated effects, have been shown to contract the pulmonary artery and the airways. In light of the observations of the instant invention these endogenous peptides may mediate various pathophysiologic states including bronchial asthma and hypoxic pulmonary vasoconstriction. Some peptides are chemotactic, e.g. eosinophil chemotactic factor, C.sub.3 a and substance P. They are generated at the site of inflammation and attract various immunological cells including neutrophils to the site. Finally, other peptides such as cholecystokinin, somatostatin, oxytocin and caerulin have potent effects on various tissues which may give rise to other pathological disorders.
Enkephalinase has been purified from kidney (Kerr, M. A. and Kenny, A. J. Biochem. J. 137: 477-488 [1974], Gafford. J. et al., Biochemistry 22, 3265-3271 [1983] and Malfroy. B. and Schwartz, J. C. Life Sci 31, 1745-1748 [1982]), intestine (Danielsen, E. M. et al., Biochem. J. 191, 545-548 [1980]), pituitary (Orlowski, M. and Wilk, S. Biochemistry 20: 4942-4945 [1981]), brain (Relton, J. M. et al., Biochem. J. 215: 755-762 [1983]) and lymph nodes (Bowes, M. A. and Kenny, A. J., Biochem. J. 236: 801-810 [1986]). Enkephalinase has been detected in many peripheral organs (Llorens. C. and Schwartz. J. C., Eur. J. Pharmacol. 69, 113-116 (1981) and in human neutrophils (Connelly, J. C. et al., Proc. Natl. Acad. Sci.[USA] 82: 8737-8741 [1985]). The distribution of enkephalinase in the brain closely parallels that of the enkephalins (Llorens, C. et al., J. Neurochem. 39: 1081-1089 [1982]). The observations of the instant invention establish that enkephalinase is also present in those peripheral tissues and cells that respond to endogenous peptides. Enkephalinase is a membrane-bound glycoprotein with subunit M.sub.r values in the range of 87,000 to 94,000. Variation in the M.sub.r values is attributed to differences in the extent and pattern of glycosylation.
The substrate specificity of enkephalinase has been studied using the enzyme from rat and human kidney. Malfroy, B. and Schwartz, J. C., J. Biol. Chem. 259: 14365-14370 (1984); Gafford et al., Biochemistry 22: 3265-3271 (1983); and Pozsgay, M. et al., Biochemistry 25: 1292-1299 (1986). These studies indicate that enkephalinase preferentially hydrolyzes peptide bonds comprising the amino group of a hydrophobic residue, shows a marked preference for short peptides, and is most efficient when it acts as a dipeptidyl carboxypeptidase releasing a carboxy terminal dipeptide. Enkephalinase, which had been found in cerebral synaptic membranes, efficiently cleaves the Gly.sup.3 -Phe.sup.4 amide bond of enkephalins (Malfroy, B. et al., Nature (Lond.) 276: 523-526 [1978]). Enkephalinase has also been found to cleave the heptapeptide (Met.sup.5)enkephalin-Arg.sup.6 -Phe.sup.7 (Schwartz, J. C. et al., In Proceedings International Union of Pharmacology 9th Congress of Pharmacology, 3: ed. by J. F. Mitchell et al., 277-283, McMillan Press Ltd., London, [1984]) as well as a variety of other neuropeptides, such as cholecystokinin (Zuzel, K. A. et al., Neuroscience 15: 149-158 [1985]), substance P (Horsthemke, B. et al. Biochem. Biophys. Res. Comm. 125: 728-733 [1984]), neurotensin (Checler et al., 1983), angiotensin I and angiotensin II (Matsas et al., Biochem J. 223 433 [1984] and Gafford et al., Biochemistry 22: 3265 [1983]), kinins, e.g. bradykinin (Gafford. J. T. et al., Biochemistry 22 3265-3271 [1983]), oxytocin (Johnson et al., 1984), and somatostatin (Mumford, R. A. et al., Proc. Natl. Acad. Sci [USA] 78:6623-6627 [1981]). While enkephalinase is capable of hydrolyzing many biological peptides in vitro (Kenny, A. J. Trends in Biochem. Sci. 11: 40-42 [1986]), in vivo enkephainase has to date only been implicated in the hydrolysis of endogenous enkephalins when released in brain (Schwartz, J. C. et al., Life Sciences 29: 1715-1740 [1981] and Lecomte, J. M. et al., J. Pharmacol Exp. Ther. 237: 937-944 [1986]). Although the levels of enkephalinase in blood are normally very low (Connelly et al., supra) enkephalinase was found to be present in high levels in the serum from patients with adult respiratory distress syndrome (Connelly et al. Supra). Enkephalinase cleaves the chemotactic tripeptide fMet-Leu-Phe. Id. It was also observed that neutrophils from donors who smoked had enkephalinase activites about twice that of nonsmokers. Id. Enkephalinase has also been found in high levels in the microvilli of human placentae (Johnson, A. R. et al., Peptides 5: 789-796 [1984]).
The present invention is based on the novel observations that specific inhibitors of enkephalinase, thiorphan, leucine-thiorphan and phosphoramidon, potentiate airway mucus secretion and smooth muscle contraction induced by endogenous peptides, e.g. substance P and other tachykinins, and kinins such as bradykinin. The invention is also based on the novel observation that in vivo enkephalinase inhibits substance P-induced increases in vascular permeability. Enkephalinase is known to cleave substance P into two fragments observed to be ineffective in stimulating mucus secretion and/or smooth muscle contraction. The invention is also based on the observation that enkephalinase digests chemotactic molecules and thus may inhibit the attraction of various inflammatory cells including neutrophils to the site of injury.
An object of the present invention is to provide a therapeutic composition for the treatment of pathological conditions in which endogenous peptides may be involved. Specifically, enkephalinase may be used as a therapeutic agent to overcome adverse effects of substance P or other neuropeptides. More specifically, enkephalinase may be used to reduce peptide-mediated mucus secretion and bronchoconstriction in the airway consequent to various diseases e.g. asthma, chronic bronchitis, cystic fibrosis, and viral infections. Enkephalinase may also be used as a therapeutic agent in the treatment of various tumors, e.g. carcinoid tumors and small cell carcinoma of the lung. Yet another object of this invention is the use of enkephalinase derivatives in the treatment of various pathological disorders mediated by certain endogenous peptides. Other peptide-induced disorders may arise in the gastrointestinal, visual, urinary, circulatory, reproductive systems and joints. The cytoplasmic and/or transmembrane deleted or substituted enkephalinase may be used in the treatment of various pathological disorders mediated by certain endogenous peptides.